This invention relates to a reaction vessel which may be used for measuring a minute amount of substance present in a living body by a simple and convenient operation.
Microanalysis of a biological substance is often carried out for the purpose of diagnosing various diseases and determining effects of various treatments. A number of assays have been developed one after another ranging from simple and convenient assays to highly sensitive assays realizing a high measurement accuracy. Among these, simplified assays, which require no measuring equipment or reaction system, are finding a wide application owing to their simple operation in such cases wherein qualitative or semi-quantitative measurements are just sufficient to make diagnoses. For example, simplified assays are used for a measurement of glucose in urine and other biochemical tests, as well as pregnancy tests. Recently, simplified assays have also been used for detection of various pathogenic viruses by nucleic acid hybridization with DNA probes.
Typical simplified assays based on immunoreactions (antigen-antibody reactions) include those utilizing an agglutination reaction (i.e. agglutination or non-agglutination) using a latex or red blood cell for their carrier and enzyme immunoassays (EIA) using an enzyme for labelling purposes.
Among the agglutination reactions, those utilizing agglutination-precipitation reaction are conducted in an ampoule having a spherical bottom surface by using red blood cell or analogous synthetic material for their carrier, and their results are evaluated by presence or size of a ring or a spot precipitated inside the spherical bottom surface. These processes may be conveniently carried out with a relatively high measurement sensitivity, but may take a long time for obtaining the results since they are based on precipitation of red blood cells or analogous synthetic materials.
The process utilizing a latex for the agglutination reaction is carried out on a slide by using a latex as their carrier. The results are evaluated after stirring the sample by observing the degree of agglutination. The latex agglutination reaction is not very sensitive, but can be carried out in a short period by a simple operation. Therefore, the latex agglutination reaction is widely employed in such application as pregnancy test wherein a high sensitivity is not necessarily required. The latex agglutination reaction, however, requires much skill for determination of the results, and therefore, those who are capable of making an accurate determination are primarily limited to doctors and laboratory technicians in medical institutions including hospitals and clinics.
The enzyme immunoassays are more sensitive than other simplified assays, but often take a relatively long reaction time for obtaining a high sensitivity. The enzyme immunoassays also suffer from a drawback that troublesome operations are required for B/F (bound/free) separation and an incomplete B/F separation would result in non-specific reactions in the subsequent enzyme reaction step leading to an erroneous evaluation of the results. B/F separation is a separation of an antigen-antibody complex (an antigen bound to an antibody, B) from free antigens or antibodies (F) in the case of an antigen-antibody reaction.
As set forth above, the simplified assays based on agglutination reactions are capable of detecting the presence of a substance, but are unsuitable for quantitative analyses wherein the amount of the substance present is to be determined. On the other hand, the enzyme immunoassays, in spite of their drawbacks of a prolonged reaction time and a troublesome B/F separation, are capable of conducting a quantitative assay as well as a qualitative assay since the results of the enzyme immunoassays may be represented in qualitative or quantitative forms by either the presence/absence or the degree of color change, namely, color development of the reaction solution. Also, the results may be easily and accurately discerned by anyone. Owing to such an advantage, a number of investigations have been carried out to shorten their reaction time and to simplify the B/F separation. As a matter of fact, an enzyme immunoassay satisfactory for practical use is not yet developed.
Recently, an assay utilizing a nucleic acid hybridization is employed for the simplified assay to detect a particular DNA or RNA (only DNA may be hereinafter mentioned, but detection of an RNA is also intended to be included within the scope of the invention). The nucleic acid-hybridization assay is analogous to the immunoassay utilizing an antigen-antibody reaction, especially an enzyme immunoassay, in that the reaction mechanism is based on selectivity of the DNA probe to hybridize with the particular DNA. Accordingly, steps included in enzyme immunoassays are likewise required in the nucleic acid-hybridization assay, and conventional nucleic acid-hybridization assays also suffer from the drawbacks of a long reaction time and a troublesome B/F separation, which should be overcome.
To overcome such drawbacks, Japanese Patent Application Kokai No. 63-20063 and Japanese Patent Application 62-215992 propose reaction vessels having a dish-like configuration.
By using the dish-like reaction vessels of these patent applications, qualitative enzyme immunoassays may be carried out by a significantly simplified procedure. These reaction vessels, however, are still insufficient to make the best of the advantage of the enzyme immunoassays that they may be used for quantitative assays.
The enzyme immunoassay involves a plurality of steps including, for example, sample dispensing and addition of washing solution, solution of an enzyme-labelled antibody, chromogenic reagent and enzyme substrate. Accordingly, this assay is quite complicated and requires a prolonged period before an evaluation can be made. These drawbacks are yet to be overcome.
For simplifying such an assay capable of conducting a quantitative evaluation, it would be essential to simplify the steps of B/F separation and addition of sample and various reagents. In such respects, the above-mentioned dish-like reaction vessels are yet to be improved.